Human NQO1 is a flavoenzyme essential for the antioxidant defense system, stabilization of tumor suppressors, and activation of quinone-based chemotherapeutics. NQO1 is over-expressed in tumors such as thyroid, breast, and lung, which makes this enzyme an attractive cancer target for drug discovery. In addition, proper NQO1 functionality and stability have been associated with other pathological situations, such as Alzheimer´s, and Parkinson´s disease. NQO1 develops its catalytic function by the “ping-pong” mechanism, which consists of two ordered steps: 1) Binding and oxidation of the electron donor NADH by FAD, reducing it to FADH2 and then NAD+ leaving the binding site; 2) Binding and reduction of a quinone substrate by FADH2. The main goal of our project will be to further understand the complex mechanism of NQO1 by determining the high-resolution crystallographic structures of the intermediates involved in the "pin-pong" mechanism by using the serial crystallography approach either at synchrotrons or at XFELs.