Structural studies of the molybdenum center of the pathogenic R160Q mutant of human sulfite oxidase by pulsed EPR spectroscopy and 17O and 33S labeling

Electron paramagnetic resonance (EPR) investigation of the Mo(V) center of the pathogenic R160Q mutant of human sulfite oxidase (hSO) confirms the presence of three distinct species whose relative abundances depend upon pH. Species 1 is exclusively present at pH < or = 6, and remains in significant amounts even at pH 8. Variable-frequency electron spin echo envelope modulation (ESEEM) studies of this species prepared with (33)S-labeled sulfite clearly show the presence of coordinated sulfate, as has previously been found for the "blocked" form of Arabidopsis thaliana at low pH (Astashkin, A. V.; Johnson-Winters, K.; Klein, E. L.; Byrne, R. S.; Hille, R.; Raitsimring, A. M.; Enemark, J. H. J. Am. Chem. Soc. 2007, 129, 14800). The ESEEM spectra of Species 1 prepared in (17)O-enriched water show both strongly and weakly magnetically coupled (17)O atoms that can be assigned to an equatorial sulfate ligand and the axial oxo ligand, respectively. The nuclear quadrupole interaction (nqi) of the axial oxo ligand is substantially stronger than those found for other oxo-Mo(V) centers studied previously. Additionally, pulsed electron-nuclear double resonance (ENDOR) measurements reveal a nearby weakly coupled exchangeable proton. The structure for Species 1 proposed from the pulsed EPR results using isotopic labeling is a six-coordinate Mo(V) center with an equatorial sulfate ligand that is hydrogen bonded to an exchangeable proton. Six-coordination is supported by the (17)O nqi parameters for the axial oxo group of the model compound, (dttd)Mo(17)O((17)Otms), where H2dttd = 2,3:8,9-dibenzo-1,4,7,10-tetrathiadecane; tms = trimethylsilyl. Reduction of R160Q to Mo(V) with Ti(III) gives primarily Species 2, another low pH form, whereas reduction with sulfite at higher pH values gives a mixture of Species 1 and 2, as well as the "primary" high pH form of wild-type SO. The occurrence of significant amounts of the "sulfate-blocked" form of R160Q (Species 1) at physiological pH suggests that this species may be a contributing factor to the lethality of this mutation.     

Resilience in reaction-diffusion systems

Reaction-diffusion systems with zero-flux Neumann boundariesare widely used to model various kinds of interaction in, forexample, the scientific fields of ecology, biology, chemistry,medicine and industry. The physical systems within these fieldsare often known to be (conditionally or unconditionally) resilientwith respect to shocks, disturbances or catastrophies in theimmediate environment. In order to be good mathematical modelsof such situations the reaction-diffusion systems must havethe same resilient or asymptotic behaviour as that of the physicalsituation. Three fundamentally different kinds of reaction termsare usually distinguished according to the entry signs of thereaction Jacobian: mutualism, mixed (predator-prey) interactionand competition. The asymptotic stability (in the Poincarésense) of mutualistic systems has already been studied extensively,but the results cannot be generalized (globally) to the othertwo fundamental types, which are not order-preserving. A partial(local) generalization is, however given here for these twotypes, involving simple Jacobian inequalities and knowledge(often prompted by the underlying physical situation) of invariantsets in solution space. The return time of resilient systemsand the approach rate of asymptotically stable solutions arealso estimated.     

Optimization of Pulsed-DEER Measurements for Gd-Based Labels: Choice of Operational Frequencies, Pulse Durations and Positions, and Temperature

In this work, the experimental conditions and parameters necessary to optimize the long-distance (≥60 Å) double electron–electron resonance (DEER) measurements of biomacromolecules labeled with Gd(III) tags are analyzed. The specific parameters discussed are the temperature, microwave band, the separation between the pumping and observation frequencies, pulse train repetition rate, pulse durations and pulse positioning in the electron paramagnetic resonance spectrum. It was found that: (1) in optimized DEER measurements, the observation pulses have to be applied at the maximum of the electron paramagnetic resonance spectrum; (2) the optimal temperature range for K_a-band measurements is 14–17 K, while in W-band the optimal temperatures are between 6 and 9 K; (iv) W-band is preferable to K_a-band for DEER measurements. Recent achievements and the conditions necessary for short-distance measurements (<15 Å) are also briefly discussed.