Structural and functional insights into a novel class of cryptochromes, the CryPro family.

Inside the vast photolyase/cryptochrome (p/c)-family Cryptochrome B of Rhodobacter sphaeroides (RsCryB) (1) is a member of a distinct enzyme class that is mainly formed by proteobacterial enzymes, therefore referred to as CryPro class. The crystal structure of RsCryB was solved by SAD-techniques at 2.7 Å resolution (2). RsCryB binds three cofactors, 6,7-dimethyl-8-ribityl-lumazine as antenna chromophore, an iron-sulfur-cluster yet unknown in the p/c-family and the catalytic FAD, bound by all characterized members (3,4). Lumazine is the second cofactor of the pterin type identified in this ancient enzyme family. EPR spectroscopy was performed on the iron-sulfur-cluster, so far identifying it as a [4Fe 4S]2+/3+ cluster, and showing an EPR characteristic akin, but not identical to characterized HiPIP-clusters (1). Spectroscopic data suggest a different photoreduction pathway as in other p/c family members, since the semiquiniod form is mostly not accumulated during photoreduction. This indicates a fast electron transfer towards the FAD after initial one electron reduction, possibly involving the Fe4S4-cluster. Mutational studies based on structural and functional data, coupled with UV/vis spectroscopy and X-ray crystallographic analysis will shed light on the functional differences between the novel CryPro type cryptochromes and already characterised enzymes of the p/c-family.