The long debated reaction mechanisms of the selective catalytic reduction (SCR) of nitric oxide with ammonia (NH3) on vanadium-based catalysts rely on the involvement of Bronsted or Lewis acid sites. This issue has been clearly elucidated using a combination of transient perturbations of the catalyst environment with operando time-resolved spectroscopy to obtain unique molecular level insights. Nitric oxide reacts predominantly with NH3 coordinated to Lewis sites on vanadia on tungsta-titania (V2O5-WO3/TiO2), while Bronsted sites are not involved in the catalytic cycle. The Lewis site is a mono-oxo vanadyl group that reduces only in the presence of both nitric oxide and NH3. We were also able to verify the formation of the nitrosamide (NH2NO) intermediate, which forms in tandem with vanadium reduction, and thus the entire mechanism of SCR. Our experimental approach, demonstrated in the specific case of SCR, promises to progress the understanding of chemical reactions of technological relevance.