Molecular dioxygen enters the active site of 12/15-lipoxygenase via dynamic oxygen access channels

Abstract

Cells contain numerous enzymes that use 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. Choosing 12/15-lipoxygenase as a typical example for such oxygen-dependent enzymes, we determined the oxygen distribution within the protein and defined potential routes for oxygen access. For this purpose, we have applied an integrated strategy of structural modeling, molecular dynamics simulations, site-directed mutagenesis, and kinetic measurements. First, we computed the 3D freeenergy distribution for oxygen, which led to identification of four oxygen channels in the protein. All channels connect the protein surface with a region of high oxygen affinity at the active site. This region is localized opposite to the nonheme iron providing a structural explanation for the reaction specificity of this lipoxygenase isoform. The catalytically most relevant path can be obstructed by L367F exchange, which leads to a strongly increased Michaelis constant for oxygen. The blocking mechanism is explained in detail by reordering the hydrogen-bonding network of water molecules. Our results provide strong evidence that the main route for oxygen access to the active site of the enzyme follows a channel formed by transiently interconnected cavities whereby the opening and closure are governed by side chain dynamics.free energy ͉ lipid peroxidation ͉ oxygen channel ͉ oxygen diffusion ͉ oxygenases

Experimental approaches to kinetics of gas diffusion in hydrogenase

“…The mutations strongly increase the Michaelis constant for H 2 , with no appreciable effect on the value of k cat (extrapolated to infinite concentration of H 2 in Table 1). This phenotype is expected when a mutation slows substrate access to the active site (4,5,8,15).…”

“…In a few cases, however, mutations of some residues located in a putative gas channel did affect the enzyme's kinetic properties. For example, certain mutations in enzymes that use molecular oxygen as substrate increase the K m for O 2 (4,5,8,15). Brzezinski and coworkers (6) demonstrated that a glycine-to-valine mutation almost completely obstructs the oxygen channel of cytochrome c oxidase; in this study, the delayed access of substrate O 2 and inhibitor CO to the active-site heme was probed by using time-resolved UV-vis spectroscopy.…”

“…As far as redox catalysis is concerned, the best-documented example is certainly the 70-Å-long channel that connects the two active sites involved in CO production and utilization in acetyl-CoA synthase/CO dehydrogenase (ACS-CODH) (2,3). In addition, channels dedicated to the transport of O 2 , N 2 , or H 2 are supposed to exist in hemecopper oxidase (4)(5)(6), nitrogenase (7), lipoxygenase (8), photosystem II (9), and both NiFe and FeFe hydrogenases (10), to cite but a few. These channels were often discovered by searching for hydrophobic cavities in x-ray structures hence their qualification as ''static.''…”

Fluorescent protein barrel fluctuations and oxygen diffusion pathways in mCherry

“…25 Recent developments in efficient computational sampling methods have allowed thorough scanning of the possible pathways for gas diffusion in the interior of proteins. [26][27][28][29] For example, such computational investigations have proved useful in understanding gas diffusion in many protein systems such as molecular dioxygen pathways via dynamic oxygen access channels in flavoproteins, [30][31][32] ammonia transport in carbamoyl phosphate synthetase, [33][34][35] and gas diffusion and channeling in hemoglobin. 28,36 In this paper, we use explicit solvent all-atom molecular dynamics (MD) simulations to investigate the protein barrel fluctuations in mCherry, which is one of the most useful monomeric variants of RFP.…”

Fungi and Their Enzymes for Pitch Control in the Pulp and Paper Industry

“…Despite extensive studies on their biochemical and molecular properties (Brash 1999;Saam et al 2007), this group of enzymes has not yet been introduced into industrial processes. Nevertheless, the specific activity of lipoxygenases to oxidize linoleic acid makes them interesting for applications in the papermaking process.…”