The dimanganese(III,IV) oxidation state of catalase from Thermus thermophilus: electron nuclear double resonance analysis of water and protein ligands in the active site

Abstract

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 mu-oxo and mu-carboxylato bridging ligands. The lack of 14N hyperfine couplings in the enzyme suggests either O6 or O5N ligand donors to each Mn. Moreover, the two sigma coordination sites on Mn(III) directed at the dz2 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 alpha and beta positions. This implicates predominantly carboxylato-type ligands (Asp and Glu) and possibly a di-mu-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(mu-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.(ABSTRACT TRUNCATED AT 400 WORDS)

Identification of the High Affinity Mn2+ Binding Site of Bacteriophage λ Phosphoprotein Phosphatase: Effects of Metal Ligand Mutations on Electron Paramagnetic Resonance Spectra and Phosphatase Activities

“…We continue to further our knowledge of why the ligand field created for the metal cluster in these hydrolytic enzymes favors hydrolytic chemistry over redox reactions, as seen in enzymes of similar active site structure, such as Mn catalases (54)(55)(56)(57). This information might lead toward the rational design of better inhibitors of this class of regulatory phosphoesterases.…”

EPR–ENDOR Characterization of (17O, 1H, 2H) Water in Manganese Catalase and Its Relevance to the Oxygen-Evolving Complex of Photosystem II

“…40, 54 The theory to explain the terminal water dipolar coupling, called extended dipole theory, incorporates the quantum mechanical projection factors for spin localized on the Mn(III) (S = 2) and Mn(IV) (S = 3/2) where these two ions are an antiferromagnetically coupled S = ½ pair. (The projection factor expresses the spin on an individual metal ion in terms of the total spin of the dimer.…”

Structure, ligands and substrate coordination of the oxygen-evolving complex of photosystem II in the S2 state: a combined EPR and DFT study

“…in Mn dimers r iso (Mn III ) = 2 and r iso (Mn IV ) = -1. Interestingly, the hyperfine and quadrupole couplings of imidazole ligands of Mn III ions differ depending on whether they represent axial (A iso = 9-13 MHz, e 2 Qq/h = 2.1-3.0 MHz) [128][129][130][131][132] or equatorial ligands (A iso = 1.5-6.6 MHz, e 2 Qq/h = 1.5-2.5 MHz). [131][132][133][134] The values seen for the His332 imino-N (A iso = 7.1 MHz, e 2 Qq/h = 1.97 MHz) fall closer to the equatorial range supporting its assignment as an equatorial ligand consistent with DFT structural models.…”