Control of the position of oxygen delivery in soybean lipoxygenase-1 by amino acid side chains within a gas migration channel

Abstract

Understanding gas migration pathways is critical to unraveling structure-function relationships in enzymes that process gaseous substrates such as O2, H2, and N2. This work investigates the role of a defined pathway for O2 in regulating the peroxidation of linoleic acid by soybean lipoxygenase 1. Computational and mutagenesis studies provide strong support for a dominant delivery channel that shuttles molecular oxygen to a specific region of the active site, thereby ensuring the regio- and stereospecificity of product. Analysis of reaction kinetics and product distribution in channel mutants also reveals a plasticity to the gas migration pathway. The findings show that a single site mutation (I553W) limits oxygen accessibility to the active site, greatly increasing the fraction of substrate that reacts with oxygen free in solution. They also show how a neighboring site mutation (L496W) can result in a redirection of oxygen toward an alternate position of the substrate, changing the regio- and stereospecificity of peroxidation. The present data indicate that modest changesina protein scaffold may modulate the access of small gaseous molecules to enzyme-bound substrates.