engleski

Non-heme iron dependent enzymes – understanding transport of iron cation in acetylacetone dioxygenase from Acinetobacter johnsoii

Diketone dioxygenase, Dke1, a functional homotetramer with Fe(II) in each monomeric subunit catalyzes O2-dependent cleavage of acetylacetone. We have studied impact of the outer shell residues on iron transport. The hydrophilic residues Glu98, Arg80 and Tyr70, which form a hydrophilic gate to the active site cavity are crucial for efficient O2 reduction. Substitution by alanine, and in the case of Glu98, glutamine, leads to a 50–100-fold decrease in O2 reduction rates. Experiments showed that these single point mutations leads to faster metal transport through Dke1, and Fe(II) depletion changes from biphasic to monophasic. In order to rationalize experiments we performed a series of molecular dynamics (MD) simulations. Simulations with Fe(II) placed in the active site revealed that the most stable is the native protein complex, while the retention time of the metal ion in the proper coordination is the shortest in the Glu98Gln variant, where, in only one of four subunits iron remained in the active site after 5 ns of MD simulations. The results enabled us to trace the possible iron ion paths and revealed the role of Glu98 in the metal ion shuttle. Finally, the simulations revealed vicinity of Glu98 and Glu11 as an alternating metal binding site.

dioxygenase; non-heme iron

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