Markov State Study of Electrostatic Channeling within the Tricarboxylic Acid Cycle Supercomplex

[Image: see text] The high efficiency of cascade reactions in supramolecular enzyme nanoassemblies, known as metabolons, has attracted substantial attention in various fields ranging from fundamental biochemistry and molecular biology to recent applications in biofuel cells, biosensors, and chemical synthesis. One reason for the high efficiency of metabolons is the structures formed by sequential enzymes that allow the direct transport of intermediates between consecutive active sites. The supercomplex of malate dehydrogenase (MDH) and citrate synthase (CS) is an ideal example of the controlled transport of intermediates via electrostatic channeling. Here, using a combination of molecular dynamics (MD) simulations and a Markov state model (MSM), we examined the transport process of the intermediate oxaloacetate (OAA) from MDH to CS. The MSM enables the identification of the dominant transport pathways of OAA from MDH to CS. Analysis of all pathways using a hub score approach reveals a small set of residues that control OAA transport. This set includes an arginine residue previously identified experimentally. MSM analysis of a mutated complex, where the identified arginine is replaced by alanine, led to a 2-fold decrease in transfer efficiency, also consistent with experimental results. This work provides a molecular-level understanding of the electrostatic channeling mechanism and will enable the further design of catalytic nanostructures utilizing electrostatic channeling.