Conformational Control Of Nitric Oxide Synthase Activity: Results From Time-Resolved And Single-Molecule Fluorescence
BIOPHYSICAL JOURNAL(2017)
Abstract
Nitric oxide synthase (NOS) generates a signaling molecule, nitric oxide, which is essential for processes such as vasodilation and neurotransmission. The enzyme forms homodimers containing cofactors FAD, FMN, and heme, each housed in subunits of the enzyme. Synthase activity in NOS is activated by calmodulin (CaM), which binds to a region adjacent to the FMN domain. Conformational motion is necessary to position sequential electron donor-acceptor pairs adjacent to one another for transfer of electrons from the FAD domain to the FMN domain and then to the oxygenase domain of the opposite member of the homodimer. We characterized the conformations and dynamics of endothelial and neuronal NOS with bound CaM by time-resolved and single-molecule detection of fluorescence from a fluorophore attached to CaM. Maximum entropy analysis of fluorescence decays for both neuronal and endothelial NOS reveals four conformational states, resolved by their distinct extents of quenching by energy transfer to heme. Clues to the identity of each population are found from their dependence on calcium concentration and from mutations known to disrupt specific interactions. The CaM mutation K115E, known to disrupt docking of CaM to the oxygenase domain, abolishes the two shortest lifetime populations. Single-molecule trajectories reveal conformational interchange on the millisecond to second time scales and permit analysis of the rates of interchange of conformational states. Exchange appears slowest to and from the conformation with highest quenching, consistent with the identification of this conformation as the “output” state of the enzyme.
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Key words
nitric oxide synthase activity,nitric oxide,fluorescence,conformational control,time-resolved,single-molecule
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