Integration of a Random‐Rapid‐Equilibrium Binding Model of NOX2 Assembly with a Five‐State Rate‐Limiting Model of NOX2 Electron Flow facilitating Superoxide Production

Abstract

Introduction Reactive oxygen species (ROS, e.g. O2.-, H2O2) play a crucial role in key physiological processes, including cell signaling. However, ROS overproduction leads to oxidative stress, which plays a critical role in cell injury/death and pathogenesis of many diseases. Mitochondria and members of the NADPH oxidase family are major sources of ROS in most mammalian cells. NOX2, the most complex and widely expressed NOX isoform, is the focus of the present study. NOX2 produces ROS, specifically, superoxide (O2.-), upon assembly of its membrane-bound subunits gp91phox and p22phox with the activated cytosolic subunits p40phox, p47phox, p67phox, and Rac. Upon NOX2 assembly and activation, electrons are transferred from substrate NADPH to molecular O2 through different redox centers of NOX2 complex, which is further regulated by pH, resulting in NADP+ and O2.-. Despite this knowledge, there is a lack of a quantitative and integrative understanding of the kinetics and regulation of NOX2 assembly and activation, electron flow, and ROS production. Method We propose four rate-limiting mechanisms for electron transfer from NADPH to O2 via NOX2 complex redox centers: 1) a five-state model with sequential electron transfer, 2) a five-state model with ping-pong electron transfer, 3) a three-state model with sequential electron transfer and full cooperativity, and 4) a three-state model with sequential electron transfer and no cooperativity. These models, which also account for pH regulation, were evaluated by fitting each to relevant experimental data. Model 1, which best described the data, was then integrated with our recent model of NOX2 assembly and activation which incorporates our hypothesized random-rapid-equilibrium binding mechanism for NOX2 assembly, regulations by guanine nucleotides (GTP, GDP), and mutual binding enhancements between individual cytosolic subunits (p40phox, p47phox, p67phox, Rac). Overall, the integrated model of NOX2 kinetics considers the thermodynamics of electron transfer through the five redox centers, biphasic inhibition by pH, assembly of cytosolic and membrane subunits, regulations by GTP and GDP, and mutual binding enhancements. Results The results of the model analysis showed that the transfer of electrons from NADPH to O2 is best described by the rate-limiting sequential electron transfer mechanism and that pH has a biphasic inhibitory effect on NOX2 reaction flux. Also, NOX2 is differentially activated and regulated by p40phox, p47phox, p67phox, and Rac subunits. The resulting integrated model can be used to quantify enhancements in the binding affinities of p47phox, p67phox and Rac subunitsby GTP, reductions in the binding affinities of the same cytosolic subunits by GDP, mutual binding enhancements between p40phox, p47phox, p67phox and Rac subunits. Conclusion We have developed and validated an integrated model of NOX2 assembly and activation, electron flow, and ROS production. The model serves as a mechanistic and quantitative framework for investigating the role of cell membrane NOX2-mediated ROS production in regulating diverse cellular mechanisms under physiological and pathophysiological conditions.