A Thermodynamically‐Constrained Mechanistic Mathematical Model for the Kinetics of NADPH Oxidase Assembly, Activation and ROS Production
Authors: Tomar Namrata , Pannala Venkat R. , Yang Chun , Cowley Allen W. , Dash Ranjan K. 
Published: November 2023

Reactive oxygen species (ROS) play an important role in immunity, cell growth, and cell signaling. However, when produced in excess, they are toxic to cells and lead to a myriad of pathologies, e.g., cardiac, renal and vascular diseases, hypertension, diabetes, and aging. One of the key producers of ROS in many cells is the family of NADPH oxidases (NOXs) on the plasma membrane. NOX is a multisubunit enzyme complex (e.g., six different subunits in NOX2), comprising of membrane and cytosolic components, which actively participates during the host cell responses to viral and bacterial infections. Of the six subunits in NOX2, gp91phox and p22phox are the integral membrane proteins that together comprise the flavocytochrome b558 (cyt b558) containing the NADPH binding site, a FAD molecule, and two molecules of heme. The other four subunits p40phox, p47phox, p67phox, and Rac2 exist in cytosol either in free form or bound together as a complex. Upon stimulation, these four subunits undergo phosphorylation, and subsequently translocate to the membrane to associate with cyt b558 to form the active enzyme leading to the transfer of electrons from NADPH to molecular oxygen for superoxide production. Although details on the enzyme structure, subunits localization, and NOXs activation are well elucidated experimentally, the kinetics of subunits assembly and their relative contributions towards NOXs activation and subsequent electron transfer reactions for ROS generation in healthy and diseased states are not clearly understood in terms of a mathematical model. In this study, we developed a thermodynamically‐constrained mechanistic mathematical model for the catalytic action of NOX2 based on available kinetic data from cell free and whole cell assay systems. We used strict thermodynamics (based on midpoint redox potentials of the electron transfer reactions) to constrain some of the model parameters and estimate the remaining parameters based on available kinetic data. The developed model is able to demonstrate the “random” binding reaction scheme for the subunits assembly and their relative contributions for NOX2 activation and electron transfer reactions for ROS production upon stimulation. Specifically, the model simulations show that: (i) the proposed mechanism is able to replicate the subunits concentration‐dependent NOX2 activation and ROS production observed in different experimental systems; (ii) the cooperative effect of p40phox on p47phox mediated NOX2 activation and ROS production; (iii) p67phox and/or Rac2 can activate cyt b558 in a catalytic manner; and (iv) the effects of various inhibitors to demonstrate the kinetics of ROS production upon NOX2 inhibition enabling detailed descriptions of their pharmacological profiles. To our knowledge, the current study represents the first a mechanism‐based kinetic model for NOX2 activation and ROS production which can reliably predict several experimental scenarios in an integrated way and is thereby of value to investigate the role of the enzyme in various pathological conditions.Support or Funding InformationNIH grants U01 HL122199 and P01 GM066730