PEF-induced structural and functional modifications of metalloenzyme SOD1 by MD simulation and in experiments

About one-quarter of all proteins use metal ions for storage, transport or enzymatic activity. We hypothesized that short electric pulses may cause direct damage of metal- binding proteins by the field forces in the extremely-high electric field. As a result of electric pulses treatment metalloproteins may release metal ion(s), experience structural rearrangements, and change functional properties. Damaging properties of short nanosecond pulsed electric field (nsPEF) has been studied using an integrated approach, combining molecular dynamics (MD) numerical modeling and experimental techniques. Superoxide dismutase molecule (SOD1) used as a model. SOD1 is 32 kDa metalloenzyme that possess copper and zinc ions and play a central role in protecting cells against reactive oxygen species. MD numerical modeling shows that intense electric field of 1,000 kV/cm given in a temporal scale of 50 ns changes the overall protein geometry. Lower field of 200 kV/cm causes distortions in SOD1 catalytic site. In experimental work, SOD1 was exposed at 200 kV/cm field given as clusters of 10 ns duration pulses. Size exclusion chromatography established no detectable aggregation, monomerization, or changes in enzyme conformation, which are indicative of absence of long term structural modifications. From the other hand, moderate 5 to10% suppression in enzyme activity was detected. The loss of activity can be related to the elimination of the catalytic copper ion from the enzyme active site as a consequence of the site distortion during nsPEF exposure. Although experimental results did not detect stable conformational modifications in SOD1, reduction of enzyme activity indicates that the induction of initial unfolding stage cannot be excluded, what is consistent with the MD numerical modeling