Pick a ska or reggae beat? Rates of conformational switching in multi‐domain flavoproteins and their relationship to electron flux through the enzymes (768.8)

Abstract

Multi‐domain enzymes rely on conformational motions to function. However, the conformational setpoints, rates of domain motions, and how they might combine to determine catalytic activity, are not well understood. To address this, we measured and compared conformational setpoints and rates of FMN domain motion in four members of the di‐flavin NADPH oxidoreductase family that catalyze important biological electron transfer reactions in mammals: cytochrome P450 reductase (CPR), methionine synthase reductase (MSR), endothelial and neuronal NO synthase (eNOS & nNOS). We used stopped flow spectroscopy, single turnover methods, and a kinetic model that relates conformational setpoint and rates of FMN domain motion to electron flux through these enzymes. Despite their highly similar protein structures and functions, the four flavoproteins displayed a surprisingly broad range of conformational setpoints (20 to 62% open state) and FMN domain motion rates (vary 100‐fold, CPR > nNOS > MSR > eNOS). Each enzyme had a unique combination of the two parameters, which in eNOS and MSR resulted in their having similar electron flux (cytochrome c reductase activity) despite having fundamentally opposite conformational behaviors (eNOS has slow conformational opening, MSR has slow conformational closing). Together, our study provides the first estimates of conformational rate parameters in this family of redox enzymes, reveals that remarkable differences exist within the family, and explains how these conformational parameters combine to determine each enzyme’s electron transfer activities.Grant Funding Source: Supported by NIH GM51491