A Well-Balanced Preexisting Equilibrium Governs Electron Flux Efficiency of a Multidomain Diflavin Reductase

Abstract

Diflavin reductases are bidomain electron transfer proteins in which structural reorientation is necessary to account for the various intramolecular and intermolecular electron transfer steps. Using small-angle x-ray scattering and nuclear magnetic resonance data, we describe the conformational free-energy landscape of the NADPH-cytochrome P450 reductase (CPR), a typical bidomain redox enzyme composed of two covalently-bound flavin domains, under various experimental conditions. The CPR enzyme exists in a salt- and pH-dependent rapid equilibrium between a previously described rigid, locked state and a newly characterized, highly flexible, unlocked state. We further establish that maximal electron flux through CPR is conditioned by adjustable stability of the locked-state domain interface under resting conditions. This is rationalized by a kinetic scheme coupling rapid conformational sampling and slow chemical reaction rates. Regulated domain interface stability associated with fast stochastic domain contacts during the catalytic cycle thus provides, to our knowledge, a new paradigm for improving our understanding of multidomain enzyme function.

Phosphorylation Controls Endothelial Nitric-oxide Synthase by Regulating Its Conformational Dynamics

“…We and others have proposed a four-state kinetic model (Fig. 1) that links the electron flux through any dual-flavin enzyme to the conformational equilibria and stochastic motions of its FMN domain (18,19). Simulations of this model have proven useful in understanding how conformational aspects regulate ET and catalysis by dual-flavin enzymes (15,16,18,20).…”

“…1) that links the electron flux through any dual-flavin enzyme to the conformational equilibria and stochastic motions of its FMN domain (18,19). Simulations of this model have proven useful in understanding how conformational aspects regulate ET and catalysis by dual-flavin enzymes (15,16,18,20).Endothelial NO synthase (eNOS) generates NO for cell signaling and functions broadly in health and disease (21-23). Its activity is regulated through several mechanisms, including post-translational modifications that in some cases may control its ET reactions.…”

Role of Protein-Protein Interactions in Metabolism: Genetics, Structure, Function

“…Beside these two residues, I245 and R246 were also recently identified, in a molecular dynamics simulation study, as two essential residues of the hinge segment, forming a part of the conformational axis and involved in a rotational movement of the FMN domain relative to the rest of the protein . S243 and R246 were also found to display large chemical shifts during the change in the conformational equilibrium between the locked and unlocked states (Frances et al, 2015). Additionally, the hinge segment was also found to be directly controlling electron transfer to cytochrome c from the reductase domain of nNOS (Haque et al, 2007(Haque et al, , 2012 as well as in CPR (Grunau et al, 2007).…”

“…conformational equilibrium between the locked and unlocked states (Frances et al, 2015). From the comparative studies of the open conformation seen in the chimeric yeast-human CPR, two residues in the hinge, G240 and S243, were proposed to be important molecular determinants for the large conformational changes .…”

Detection of domain motion in NADPH-cytochrome P450 oxidoreductase through polarization anisotropy measurements

“…Because the complex of POR with cytochrome P450 has never been structurally resolved, how these conformational states of POR relate to the steps in the functional cycle-remains debated. For example, oxidized POR has been demonstrated to assume closed conformation (blue ribbon in Figure 1) in solution by NMR spectroscopy and small-angle X-ray scattering [5][6][7] , while other reports indicated it is in an open form in the oxidized state 8,9 . In this study, we probed conformational states of the oxidized cytosolic fragment of POR, residues 57-678, (in the following text-POR) from viewpoint of the local conformational freedom measured by rotational diffusion constants.…”