Flavoprotein Protocols

Methods in Molecular Biology TM VOLUME 131 Flavoprotein Protocols Edited by Stephen K. Chapman Graeme A. Reid HUMANA PRESS UV-Visible Spectroscopy 1 1 UV-Visible Spectroscopy as a Tool to Study Flavoproteins Peter Macheroux 1. Introduction The essential cofactor of flavoproteins is derived from riboflavin (Fig. 1), which consists of the isoalloxazine ring system with a ribityl side chain attached to the central N-10 position in the pyrazine moiety. This precursor is phosphorylated at the 5'-hydroxyl group by flavokinase to yield flavin mononucleotide (FMN). In a second ATP-dependent reaction, FAD pyrophosphorylase attaches an AMP moiety to FMN yielding flavin adenine dinucleotide (FAD). Both cofactors are widely distributed in nature. The side chain plays an important role in binding of the flavin ring system to the protein and therefore the majority of flavoproteins have a strong preference for either FMN or FAD. On the other hand, riboflavin does not play a general role as a cofactor in flavoenzymes. In addition to the noncovalent binding of the flavin to the protein, covalent linkage to an amino acid residue via the 8- or 6-position of the isoalloxazine ring system is also found (see Chapter 13). In all cases, the mechanistically relevant moiety is the isoalloxazine ring system that serves as a reversible reduction-oxidation catalyst in many biological redox processes (for a review see: (1,2)). This is also the structural component of the flavin cofactor that is responsible for light absorption in the UV and visible spectral range giving rise to the yellow appearance of the flavin and of flavoproteins (Latin: flavus = yell