Kurt Warncke

A hydrogen-atom abstraction model for the function of YZ in photosynthetic oxygen evolution

Recent magnetic-resonance work on YŻ suggests that this species exhibits considerable motional flexibility in its functional site and that its phenol oxygen is not involved in a well-ordered hydrogen-bond interaction (Tang et al., submitted; Tommos et al., in press). Both of these observations are inconsistent with a simple electron-transfer function for this radical in photosynthetic water oxidation. By considering the roles of catalytically active amino acid radicals in other enzymes and recent data on the water-oxidation process in Photosystem II, we rationalize these observations by suggesting that YŻ functions to abstract hydrogen atoms from aquo- and hydroxy-bound managanese ions in the (Mn)4 cluster on each S-state transition. The hydrogen-atom abstraction process may occur either by sequential or concerted kinetic pathways. Within this model, the (Mn)4/YZ center forms a single catalytic center that comprises the Oxygen Evolving Complex in Photosystem II.

Engineering metal ion coordination to regulate amyloid fibril assembly and toxicity

Protein and peptide assembly into amyloid has been implicated in functions that range from beneficial epigenetic controls to pathological etiologies. However, the exact structures of the assemblies that regulate biological activity remain poorly defined. We have previously used Zn2+ to modulate the assembly kinetics and morphology of congeners of the amyloid β peptide (Aβ) associated with Alzheimers disease. We now reveal a correlation among Aβ-Cu2+ coordination, peptide self-assembly, and neuronal viability. By using the central segment of Aβ, HHQKLVFFA or Aβ(13–21), which contains residues H13 and H14 implicated in Aβ-metal ion binding, we show that Cu2+ forms complexes with Aβ(13–21) and its K16A mutant and that the complexes, which do not self-assemble into fibrils, have structures similar to those found for the human prion protein, PrP. N-terminal acetylation and H14A substitution, Ac-Aβ(13–21)H14A, alters metal coordination, allowing Cu2+ to accelerate assembly into neurotoxic fibrils. These results establish that the N-terminal region of Aβ can access different metal-ion-coordination environments and that different complexes can lead to profound changes in Aβ self-assembly kinetics, morphology, and toxicity. Related metal-ion coordination may be critical to the etiology of other neurodegenerative diseases.

Local Structure and Global Patterning of Cu2+ Binding in Fibrillar Amyloid-β [Aβ(1–40)] Protein

The amyloid-β (Aβ) protein forms fibrils and higher-order plaque aggegrates in Alzheimers disease (AD) brain. The copper ion, Cu(2+), is found at high concentrations in plaques, but its role in AD etiology is unclear. We use high-resolution pulsed electron paramagnetic resonance spectroscopy to characterize the coordination structure of Cu(2+) in the fibrillar form of full-length Aβ(1-40). The results reveal a bis-cis-histidine (His) equatorial Cu(2+) coordination geometry and participation of all three N-terminal His residues in Cu(2+) binding. A model is proposed in which Cu(2+)-His6/His13 and Cu(2+)-His6/His14 sites alternate along the fibril axis on opposite sides of the β-sheet fibril structure. The local intra-β-strand coordination structure is not conducive to Cu(2+)/Cu(+) redox-linked coordination changes, and the global arrangement of Cu sites precludes facile multielectron and bridged-metal site reactivity. This indicates that the fibrillar form of Aβ suppresses Cu redox cycling and reactive oxygen species production. The configuration suggests application of Cu(2+)-Aβ fibrils as an amyloid architecture for switchable electron charge/spin coupling and redox reactivity.

Comparative model of EutB from coenzyme B12‐dependent ethanolamine ammonia‐lyase reveals a β8α8, TIM‐barrel fold and radical catalytic site structural features

The structure of the EutB protein from Salmonella typhimurium, which contains the active site of the coenzyme B12 (adenosylcobalamin)‐dependent enzyme, ethanolamine ammonia‐lyase, has been predicted by using structural proteomics techniques of comparative modelling. The 453‐residue EutB protein displays no significant sequence identity with proteins of known structure. Therefore, secondary structure prediction and fold recognition algorithms were used to identify templates. Multiple three‐dimensional template matching (threading) servers identified predominantly β8α8, TIM‐barrel proteins, and in particular, the large subunits of diol dehydratase (PDB: 1eex:A, 1dio:A) and glycerol dehydratase (PDB: 1mmf:A), as templates. Consistent with this identification, the dehydratases are, like ethanolamine ammonia‐lyase, Class II coenzyme B12‐dependent enzymes. Model building was performed by using MODELLER. Models were evaluated by using different programs, including PROCHECK and VERIFY3D. The results identify a β8α8, TIM‐barrel fold for EutB. The β8α8, TIM‐barrel fold is consistent with a central role of the α/β‐barrel structures in radical catalysis conducted by the coenzyme B12‐ and S‐adenosylmethionine‐dependent (radical SAM) enzyme superfamilies. The EutB model and multiple sequence alignment among ethanolamine ammonia‐lyase, diol dehydratase, and glycerol dehydratase from different species reveal the following protein structural features: (1) a “cap” loop segment that closes the N‐terminal region of the barrel, (2) a common cobalamin cofactor binding topography at the C‐terminal region of the barrel, and (3) a β‐barrel‐internal guanidinium group from EutB R160 that overlaps the position of the active‐site potassium ion found in the dehydratases. R160 is proposed to have a role in substrate binding and radical catalysis. Proteins 2006.

OPTESIM, a Versatile Toolbox for Numerical Simulation of Electron Spin Echo Envelope Modulation (ESEEM) that Features Hybrid Optimization and Statistical Assessment of Parameters

Electron spin echo envelope modulation (ESEEM) is a technique of pulsed-electron paramagnetic resonance (EPR) spectroscopy. The analyis of ESEEM data to extract information about the nuclear and electronic structure of a disordered (powder) paramagnetic system requires accurate and efficient numerical simulations. A single coupled nucleus of known nuclear g value (g(N)) and spin I=1 can have up to eight adjustable parameters in the nuclear part of the spin Hamiltonian. We have developed OPTESIM, an ESEEM simulation toolbox, for automated numerical simulation of powder two- and three-pulse one-dimensional ESEEM for arbitrary number (N) and type (I, g(N)) of coupled nuclei, and arbitrary mutual orientations of the hyperfine tensor principal axis systems for N>1. OPTESIM is based in the Matlab environment, and includes the following features: (1) a fast algorithm for translation of the spin Hamiltonian into simulated ESEEM, (2) different optimization methods that can be hybridized to achieve an efficient coarse-to-fine grained search of the parameter space and convergence to a global minimum, (3) statistical analysis of the simulation parameters, which allows the identification of simultaneous confidence regions at specific confidence levels. OPTESIM also includes a geometry-preserving spherical averaging algorithm as default for N>1, and global optimization over multiple experimental conditions, such as the dephasing time (tau) for three-pulse ESEEM, and external magnetic field values. Application examples for simulation of (14)N coupling (N=1, N=2) in biological and chemical model paramagnets are included. Automated, optimized simulations by using OPTESIM lead to a convergence on dramatically shorter time scales, relative to manual simulations.

Copper(II)-bis-Histidine Coordination Structure in a Fibrillar Amyloid β-Peptide Fragment and Model Complexes Revealed by Electron Spin Echo Envelope Modulation Spectroscopy

Truncated and mutated amyloid‐β (Aβ) peptides are models for systematic study—in homogeneous preparations—of the molecular origins of metal ion effects on Aβ aggregation rates, types of aggregate structures formed, and cytotoxicity. The 3D geometry of bis‐histidine imidazole coordination of CuII in fibrils of the nonapetide acetyl‐Aβ(13–21)H14A has been determined by powder 14N electron spin echo envelope modulation (ESEEM) spectroscopy. The method of simulation of the anisotropic combination modulation is described and benchmarked for a CuII‐bis‐cis‐imidazole complex of known structure. The revealed bis‐cis coordination mode, and the mutual orientation of the imidazole rings, for CuII in Ac‐Aβ(13–21)H14A fibrils are consistent with the proposed β‐sheet structural model and pairwise peptide interaction with CuII, with an alternating [‐metal‐vacancy‐]n pattern, along the N‐terminal edge. Metal coordination does not significantly distort the intra‐β‐strand peptide interactions, which provides a possible explanation for the acceleration of Ac‐Aβ(13–21)H14A fibrillization by CuII, through stabilization of the associated state and low‐reorganization integration of β‐strand peptide pair precursors.

Analysis of static distributions in hydrogen hyperfine interactions in randomly oriented radicals in the solid state by using 2H electron spin echo envelope modulation spectroscopy: Conformational dispersion of β ‐2H coupling in the model tyrosyl radical

The experimental analysis of static distributions in hydrogen hyperfine interactions in randomly‐oriented organic radicals in the solid state by using 2H electron spin echo envelope modulation spectroscopic techniques has been examined systematically. The hyperfine interaction between the two β‐methylene‐2H nuclei and coupling π‐spin density (ρπ) at ring carbon atom C1 in the tyrosine neutral radical trapped in a low temperature aqueous glass was addressed specifically. Stimulated echo envelope modulation generated by the microwave pulse‐swapping sequence was collected for τ values of 176–1295 ns at external magnetic field strengths of 0.3258 and 0.3983 T. The spectra reveal weak (β‐2Hw) and strong (β‐2Hs) sets of hyperfine couplings. The envelope modulation depths and line shape responses to changes in τ and magnetic field strength could not be reproduced by simulations that incorporated discrete principal hyperfine tensors. Successful simulations were achieved by using two sets of distributed principal ...

Reaction of the CoII-Substrate Radical Pair Catalytic Intermediate in Coenzyme B12-Dependent Ethanolamine Ammonia-Lyase in Frozen Aqueous Solution from 190 to 217 K☆

The decay kinetics of the aminoethanol-generated Co(II)-substrate radical pair catalytic intermediate in ethanolamine ammonia-lyase from Salmonella typhimurium have been measured on timescales of <10(5) s in frozen aqueous solution from 190 to 217 K. X-band continuous-wave electron paramagnetic resonance (EPR) spectroscopy of the disordered samples has been used to continuously monitor the full radical pair EPR spectrum during progress of the decay after temperature step reaction initiation. The decay to a diamagnetic state is complete and no paramagnetic intermediate states are detected. The decay exhibits three kinetic regimes in the measured temperature range, as follows. i), Low temperature range, 190 < or = T < or = 207 K: the decay is biexponential with constant fast (0.57 +/- 0.04) and slow (0.43 +/- 0.04) phase amplitudes. ii), Transition temperature range, 207 < T < 214 K: the amplitude of the slow phase decreases to zero with a compensatory rise in the fast phase amplitude, with increasing temperature. iii), High temperature range, T > or = 214 K: the decay is monoexponential. The observed first-order rate constants for the monoexponential (k(obs,m)) and the fast phase of the biexponential decay (k(obs,f)) adhere to the same linear relation on an lnk versus T(-1) (Arrhenius) plot. Thus, k(obs,m) and k(obs,f) correspond to the same apparent Arrhenius prefactor and activation energy (logA(app,f) (s(-1)) = 13.0, E(a,app,f) = 15.0 kcal/mol), and therefore, a common decay mechanism. We propose that k(obs,m) and k(obs,f) represent the native, forward reaction of the substrate through the radical rearrangement step. The slow phase rate constant (k(obs,s)) for 190 < or = T < or = 207 K obeys a different linear Arrhenius relation (logA(app,s) (s(-1)) = 13.9, E(a,app,s) = 16.6 kcal/mol). In the transition temperature range, k(obs,s) displays a super-Arrhenius increase with increasing temperature. The change in E(a,app,s) with temperature and the narrow range over which it occurs suggest an origin in a liquid/glass or dynamical transition. A discontinuity in the activation barrier for the chemical reaction is not expected in the transition temperature range. Therefore, the transition arises from a change in the properties of the protein. We propose that a protein dynamical contribution to the reaction, which is present above the transition temperature, is lost below the transition temperature, owing to an increase in the activation energy barrier for protein motions that are coupled to the reaction. For both the fast and slow phases of the low temperature decay, the dynamical transition in protein motions that are obligatorily coupled to the reaction of the Co(II)-substrate radical pair lies below 190 K.

Redox state dependence of rotamer distributions in tyrosine and neutral tyrosyl radical.

Redox state-dependent changes in the relative orientation of the phenol side chain and the peptide group in model tyrosine have been characterized using specific 2H isotopic labelling and X-band electron paramagnetic resonance (EPR) spectroscopy. Tyrosyl radicals were generated by UV photolysis of tyrosine trapped in rigid polycrystalline basic-aqueous medium at T < or = 170 K. Ring-2H(4) and beta-2H(2) substitutions on tyrosine were used to enhance the lineshape contributions from beta-hydrogen or ring-hydrogen hyperfine interactions, respectively. The EPR lineshape at 120 K of the trapped ring-2H(4)-tyrosyl radical is altered dramatically after annealing at 235 K. In contrast, the lineshape of the beta-2H(2)-tyrosyl radical is impervious to annealing. The effect of annealing on the lineshape therefore arises from a change in the isotropic hyperfine coupling between unpaired pi-electron spin density at the ring carbon atom C(1) and the beta-hydrogen nuclei, which is caused by rotational relaxation of the ring and peptide group about the C(1)-C(beta) bond. EPR simulations indicate angular distributions of the peptide group (R-) of 0 degrees < or = theta(R) < or = 30 degrees and 0 degrees < or = theta(R)< or = 18 degrees in the rigid and relaxed radical states, respectively. Redox-induced changes in the C(1)-C(beta) rotamer distribution must be accounted for in assessments of stable amino acid side chain equilibrium structures, and may influence catalytic tyrosyl radical/tyrosine function in enzymes.

2H electron spin echo envelope modulation spectroscopy of strong, α‐hydrogen hyperfine coupling in randomly oriented paramagnetic systems

Experimental characterization of strong, anisotropic 2H hyperfine interactions in randomly oriented organic radicals in the solid state by using electron spin echo envelope modulation (ESEEM) spectroscopic techniques has been examined systematically. The α‐3,5‐2H coupling in the tyrosine neutral radical in a low temperature aqueous glass was used as a model. Envelope modulation was obtained by integration of the stimulated‐echo generated by a three‐pulse, microwave pulse‐swapping sequence. Division of envelope modulation from the 2H‐substituted radical by that from the per‐protonated radical remedies discontinuities introduced in the envelope by the eclipse of the second and third pulses. Envelope modulation data was collected for τ values from 214 to 1295 ns (9.132 GHz, 0.3265 T). The common, spectrometer dead‐time‐limited data collection start‐point (140 ns) for the different τ values attenuates loss of anisotropic information and allows summation of time domain data. The Fourier transform of the envelo...