Molecular Dynamics of Oxygen Transport in Lipoygenase

Molecular Dynamics Simulations (MD) provide valuable information about dynamics and energetics of molecular structures especially where experimental detail is missing (more about MD in general and Paratool).

Oxygen diffusion Cells contain a variety of oxygenating enzymes with a more or less well known mechanism of catalysis. The way oxygen is taking to the active center has only been studied in detail for a few years in a handful of proteins although this question is crucial for an understanding of the reaction on a stuctural level.

In some proteins, channels exist serving to absorb oxygen from the solvent and leading it to the catalytic center. We are investigating oxygen transport properties of several oxygenases using MD simulations. Primary results suggest that such an oxygen tunnel is also present in 15-lipoxygenase (LOX). This hypothesis is currently beeing validated.

Lipoxygenase Reaction

Lipoxygenases catalyze the position- and stereospecific dioxygenation of fatty acids and lipids to chiral conjugated hydroperoxy compounds. They are found in all higher organisms and their products are the precursors of a number of physiological effectors. Some details of the catalytic mechanisms of these enzymes are still unknown. In particular, it is unclear how molecular oxygen penetrates deep into the binding pocket and how the fatty acid substrate is aligned to the active site containing an octahedral iron complex. The ligands of the iron cluster in 15-lipoxygenase (LOX) [1] are constituted by four histidines, the C-terminus of the protein chain and a hydroxy anion pointing towards the substrate (Fig. 1). During the reaction the hydroxide abstracts a proton from the fatty acid while the Fe3+ is reduced to Fe2+ taking up one electron [2]. Then the resulting lipid radical reacts with dioxygen to form the peroxide.

Determination of Force Field Parameters

In order to perform molecular dynamics simulations, force field parameters for the six-coordinated iron complex have to be determined. For the following ab initio calculations, a simplified cluster model following the principles shown in [2] was used: The histidines are replaced by ammonia, the C-terminal carboxyl group was modeled as formiate, while the sixth ligand is the hydroxy anion (Fig. 1). The molecular geometry was optimized employing density functional theory calculations.

Fig. 1: Iron Cluster. (a) The ligands are constituted by four histidine imidazole rings, the C-terminus (behind Fe) and a hydroxy anion (left). (b) Simplified cluster model.

The Hessian matrix consists of the second derivatives of the potential energy with respect to the coordinates. Assuming a locally harmonic potential surface at the equilibrium, the eigenvalues of the Hessian represent the squares of the normal mode frequencies of vibration. These were projected onto the internal coordinates such as bonds and angles and the force constants determined from the according frequencies.

Fig. 2: Iron d-orbital.
The partial charges for the atoms were obtained using electrostatic potential fitting (ESP) where the set of atom based point charges is searched, whose electrostatic potential fits best the one arising from the electron density distribution at the equilibrium geometry. The sum of the partial charges must of course equal the net charge of the molecule.

Interestingly, modeling the cluster based on electrostatic terms only does not yield a stable octahedral geometry. Therefore, electronic effects like bonded interaction of the ligand electrons with the d-orbitals of iron and the partial charge transfer from the ligands in to the empty iron 4s orbital have to be taken into acccount (Figs. 2, 3). They contribute mainly to the bond and angle terms of the force field.

Fig. 3: (a) Iron 4s-orbital (partially populated by ligand electrons). (b) Natural localized molecular orbital showing electron density shifted from ligand to iron.

Investigation of Oxygen Transport Processes

The importance of protein dynamics for oxygen transport is clearly shown by the multiple conformation crystal structure of cholesterol oxidase containing more than one conformation for many of the sidechains [3]. In the conformation with the highest electron density no channel is found while in another conformation a long tunnel into the active side exists.

Fig. 4: Arachidonic acid in binding pocket.
We employed MD simulations to reveal the dynamic features of LOX and to determine the oxygen pathway. In different simulations, oxygen was inserted at the catalytic site and in the solvent and its diffusion in the protein matrix was studied. Similar to the situation in cholesterol oxydase, our results show that there is a specific oxygen channel apart from the substrate entrance leading from the solvent phase to the active site. In LOX, the channel cannot be seen in the crystal structure, nevertheless its different segments open temporarily during the simulation allowing oxygen molecules to diffuse into the protein. The interior end of the tunnel represents the ideal point for the correct stereo- and position specific insertion of dioxygen into the substrate.


Researchers

Dr. Jan Saam
Prof. Hermann-Georg Holzhütter

References

[1] Gillmor SA, Villaseņor A, Fletterick R, Sigal E, Browner MF. (1997) The Structure of mammalian 15-lipoxygenase reveals similarity to the lipases and the determinants of substrate specificity. Nat. Struct. Biol., 4, 1003-1009 [PubMed]

[2] Lehnert N, Solomon EI. (2003) Density-functional investigation on the mechanism of H-atom abstraction by lipoxygenase. J. Biol. Inorg. Chem., 8, 294-305. [PubMed]

[3] Lario PI, Sampson N, Vrielink A. (2003) Sub-atomic resolution crystal structure of cholesterol oxidase: What atomic resolution crystallography reveals about enzyme mechanism and the role of FAD cofactor in redox activity. J. Mol. Biol., 326, 1635-1650. [PubMed]

Own Publications

Saam J, Ivanov I, Walther M, Holzhütter H, and Kuhn H. (2007) Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels. Proc. Natl. Acad. Sci., 104(33), 13319-13324 [PubMed]

Ivanov I, Saam J, Kühn H, Holzhütter HG. (2005) Dual role of oxygen during lipoxygenase reactions. FEBS Journal, 272, 2523-2535. [PubMed, pdf]

Kühn H, Saam J, Eibach S, Holzhütter HG, Ivanov I, Walther M. (2005) Structural biology of mammalian lipoxygenases: Enzymatic consequences of targeted alterations of the protein structure. Biochem. Biophys. Res. Commun., 338, 93-101. [PubMed]