Single-Molecule Spectroscopy Reveals How Conformational Ensembles Modulate an Enzymatic Electron Transfer Relay

Iris Grossman, Structural Biology, Weizmann Institute of Science, Rehovot, Israel

The ability to query enzyme molecules individually is transforming our view of catalytic mechanisms. Quiescin sulfhydryl oxidase (QSOX) is a Golgi-localized and secreted catalyst of disulfide bond formation that relays electrons from substrate cysteines via multiple redox-active sites to molecular oxygen. These redox-active sites are located on two different protein domains tethered by a flexible linker, enabling them to work both in cooperation and independently. Single-molecule Förster resonance energy transfer (FRET) revealed two distinct conformational populations in the resting enzyme, a minor population in which the domains closely interact (closed state), and a larger population in which the domains tumble freely (open state).  Initiating the electron relay strikingly altered the occupancy of these two populations. Specifically, a correlation was found between turnover and the amount of closed state in a substrate concentration range in which electron supply was limiting. However, the opposite trend was observed when the system was supplied with an excess of electrons. During the development of an expanded model for the QSOX catalytic mechanism-- required to reconcile single-molecule and bulk turnover measurements-- a new appreciation was gained for the role of enzyme conformational states in partitioning between competing electron transfer routes. More generally, the new model demonstrated how the phenomena of substrate activation and inhibition can arise naturally from flux among conformational and chemical states. These studies illustrate the use of gross spectroscopic handles to illuminate the fine structure of enzyme activity.