Identification of dynamic oxygen access channels in 12/15-lipoxygenase

Cells contain numerous enzymes utilizing molecular oxygen for their reactions. Often, their active sites are buried deeply inside the protein which raises the question whether there are specific access channels guiding oxygen to the site of catalysis. In the present thesis this question is addressed choosing 12/15lipoxygenase as a typical example for such oxygen dependent enzymes. Lipoxygenases are found in all higher organisms and their products are the precursors of a number of physiological effectors such as inflammation mediators or hormones. They catalyze the positionand stereospecific dioxygenation of fatty acids and lipids to chiral conjugated hydroperoxy compounds. Directed oxygen access to the reaction site through a channel would explain important aspects of the enzyme’s stereochemical specificity. The oxygen distribution within the protein was determined and potential routes for oxygen access were defined. For this purpose an integrated strategy of theoretical and experimental studies including structural modeling, molecular dynamics simulations, site directed mutagenesis, and kinetic measurements was applied. The limited scope of currently available force fields for biomolecular simulations required the development of force field parameters for the nonheme iron complex in lipoxygenase. The missing parameters were determined based on density functional theory calculations of a simplified model of the coordination complex. A series of molecular dynamics simulations of the protein in solution was performed. From the trajectories, the 3-dimensional distribution of the freeenergy cost for placing oxygen at a certain position could be computed by means of the implicit ligand sampling algorithm. Analyzing energetically favorable paths in the free-energy map led to identification of four oxygen channels in the protein. All channels connect the protein surface with a zone of high oxygen affinity at the active site. This region is localized opposite to the non-heme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. Furthermore, it can be seen that the oxygen occupation probability around atom C15 of the arachidonic acid backbone is 7 fold higher than around C11. Thus oxygen insertion at C15 appears to be favored over C11, which is consistent with the positional specificity of the enzyme. However, owing to the high degree of structural flexibility of arachidonic acid the calculations do not exclude oxygen insertion at C11 or at the pro-R side of C15 so that additional mechanisms may contribute to determine the stereochemistry of the oxygenation process. The catalytically most relevant path can be obstructed by L367F exchange which leads to a strongly increased Michaelis constant for oxygen. This experimentally proven blocking mechanism can, by virtue of molecular dynamics studies of the mutated protein, be explained in detail through a reordering of the hydrogen bonding network of water molecules inside the protein. As a conclusion, the results of this thesis and the related experiments provide strong evidence that specialized oxygen access channels exist. The main route for oxygen access to the active site of 12/15-lipoxygenase is formed of neighboring, mostly hydrophobic cavities which partition oxygen away from water. These cavities are transiently interconnected due to amino acid side chain flexibility.