Quiescin sulfhydryl oxidase (QSOX) is a Golgi-localized and secreted catalyst of disulfide bond formation that relays electrons from substrate cysteines to molecular oxygen. QSOX exhibits large-scale domain dynamics and an interdomain dithiol/disulfide electron-transfer step in its catalytic cycle. The ability to query enzyme molecules individually is transforming our view into catalytic mechanisms, inspiring us to apply single-molecule fluorescence resonance energy transfer (FRET) to the QSOX catalytic cycle. These studies revealed the distribution of conformational and chemical states under various reaction conditions. In particular, the distinct signature of the interdomain electron-transfer intermediate on the single-molecule level was used to resolve an outstanding ambiguity in the QSOX mechanism. Further investigation into the requirements for population of this intermediate led to discovery of a substrate-activation process that enhances enzyme turnover by blocking electron backflow. A combination of single-molecule experiments together with bulk turnover assays preformed on various QSOX mutants allowed specific steps in the catalytic cycle to be probed individually. The distribution of enzyme conformational populations and the dependence of overall turnover number on substrate concentration were modulated in striking ways by mutations that affected various rate constants within the multi-step mechanism. These studies show how gross spectroscopic handles can provide information not only on protein conformational changes but also on intimate chemical steps in enzyme mechanisms.