The 1H hyperfine tensors of the dimanganese(III,IV) oxidation state of the non-heme-type catalase enzyme from the thermophilic bacterium Thermus thermophilus have been measured by electron nuclear double resonance (ENDOR) spectroscopy at pH 6.5–9. These were compared to model dimanganese-(III,IV) complexes possessing six-coordinate N4O2, N3O3, and O6 atom donor sets to each Mn and µ-oxo and µ-carboxylato bridging ligands. The lack of 14N hyperfine couplings in the enzyme suggests either or O5N ligand donors to each Mn. Moreover, the two σ coordination sites on Mn(III) directed at the [formula omitted] orbital cannot be occupied by N ligands. The 1H ENDOR spectrum revealed two types of anisotropic tensors, attributable to two D2O-exchangeable protons on the basis of the magnitude of the electron paramagnetic resonance (EPR) line narrowing in D2O. All six of the 1H hyperfine couplings are proposed to arise from a single displaceable water molecule in the active site, on the basis of their reversible disappearance, upon incubation in D2O or by precipitation from ammonium sulfate, and by simulation of the 1H ENDOR spectrum. The Mn ions are coordinated predominantly by nonmagnetic O atoms lacking covalently bound protons in both α and β positions. This implicates predominantly carboxylato-type ligands (Asp and Glu) and possibly a di-µ-oxo bridge between Mn ions. The latter is supported also by the presence of strong antiferromagnetic coupling. Comparison to other dimetalloproteins also possessing the four-helix bundle structural motif shows that the polyoxo(carboxylato) coordination in catalase differs significantly from the polyhistidine coordination adopted by the diiron(II,II) site in the O2-binding protein myohemerythrin, but resembles the polycarboxylato ligation adopted by the diiron(III,III) site of ribonucleotide reductase. The catalase 1H ENDOR spectrum is essentially identical to that for the exchangeable protons in the active site of the diiron(II,III) state of uteroferrin, an acid phosphatase [Doi et al. (1988) J. Biol. Chem. 263, 5757–5763], and also for a polycarboxylato complex possessing the Mn2(µ-O)2 core with H-bonded water ligands. The 1H ENDOR line shape in catalase could be simulated using a theoretical model suitable for multispin clusters. It treats the two Mn spins as point dipoles which are exchange-coupled. It includes both dipolar and isotropic ligand hyperfine couplings. Using this model, the position of the proton with the largest interaction could be located with respect to the Mn-Mn vector because of the extreme sensitivity of line shape to position. Four possible positions are predicted, differing solely by the undetermined sign of the experimental hyperfine tensor elements. Analysis of the 1H ENDOR spectrum via angle selection using the EPR line shape anisotropy enabled determination of the location of the proton relative to the [formula omitted] orbital of Mn(III), which is orthogonal to the presumed Mn2(µ-O)2 plane. Simulation of the angle-selected 1H ENDOR spectrum revealed that only one proton position could account for the spectrum. This gives distances of 4.6 and 2.9 Å to Mn(III) and Mn(IV), respectively. These coordinates are compatible with a water or hydroxide molecule H-bonded to a terminal ligand on Mn(IV). A second proton exists at a greater distance. The structural basis for the lack of catalase activity in the Mn2(III,IV) oxidation state is suggested to originate from the kinetic inertness to substitution or reduction of the bridging oxo atoms in the Mn2(µ-O)2 core.
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