The key role of water in the dioxygenase function of Escherichia coli flavohemoglobin

Flavohemoglobins (FHbs) are members of the globin superfamily, widely distributed among prokaryotes and eukaryotes that have been shown to carry out nitric oxide dioxygenase (NOD) activity. In prokaryotes, such as Escherichia coli, NOD activity is a defence mechanism against the NO release by the macrophages of the hosts' immune system during infection. Because of that, FHbs have been studied thoroughly and several drugs have been developed in an effort to fight infectious processes. Nevertheless, the protein's structural determinants involved in the NOD activity are still poorly understood. In this context, the aim of the present work is to unravel the molecular basis of FHbs structural dynamics-to-function relationship using state of the art computer simulation tools. In an effort to fulfill this goal, we studied three key processes that determine NOD activity, namely i) ligand migration into the active site ii) stabilization of the coordinated oxygen and iii) intra-protein electron transfer (ET). Our results allowed us to determine key factors related to all three processes like the presence of a long hydrophobic tunnel for ligand migration, the presence of a water mediated hydrogen bond to stabilize the coordinated oxygen and therefore achieve a high affinity, and the best possible ET paths between the FAD and the heme, where water molecules play an important role. Taken together the presented results close an important gap in our understanding of the wide and diverse globin structural-functional relationships.

Flavohemoglobin dioxygenase function as studied in the present work, relies on ligand (oxygen and nitric oxide) migration to the active site (left panel), chemical reaction between NO and oxy-heme to yield nitrate (middle panel) and heme re-reduction through intramolecular electron transfer (right panel).Figure optionsDownload as PowerPoint slideHighlights
► Small ligand (O2, NO) migration into the heme active site was studied.
► Stabilization of the coordinated oxygen and intra-protein ET was analyzed.
► A long hydrophobic tunnel is present for ligand migration.
► The presence of a water mediated hydrogen bond stabilizes the coordinated oxygen.
► The best possible ET paths from FAD to heme go through a water and heme propionates.