Anaerobic stopped-flow (SF) spectrophotometry is a powerful biophysical tool that allows a complete kinetic characterization of protein interactions with other molecules when they are in different redox states, as well as of the redox processes consequence of such interactions. Differences in the absorption spectroscopic properties of oxidized, semiquinone and hydroquinone states of flavoproteins, as well as the appearance of transient spectroscopic features produced by the flavin cofactor during substrate binding and electron transfer processes, have made SF a suitable technique for kinetically dissecting their mechanisms of reaction. In addition, SF coupled to photodiode array detection, enables kinetic data collection in a wavelength range. When such type of data are available for a flavoprotein reaction, they allow for obtaining detailed information of individual reaction steps, including intermolecular dissociation constants as well as electron transfer rate constants. Methodologies for the mechanistic characterization of flavoproteins involved in redox processes by SF spectrophotometry are described in this chapter.
Keywords: Binding; Electron transfer; Fast kinetics; Flavoprotein oxidation; Flavoprotein reduction; Photodiode array detection; Stopped-flow.