The Function of the Halophilic Dodecin

Flavins are physiologically relevant cofactors that catalyze
various redox and light-induced reactions. Due to a high intrinsic
reactivity, these compounds are found tightly bound to proteins
with the chemistry of the flavin either narrowed to a defined
reaction channel (flavoenzymes) or reduced to (almost)
non-reactivity (flavin binding and carrier proteins). Lumichrome is
a product of flavin photodegradation. In spite of the structural
similarity to flavins, lumichrome has electronic properties which
differ from flavins, preventing this compound from any
physiological relevance as a cofactor. The interest in lumichrome
is basically focussed on its role as a photosensitizing compound.
Lumichrome is excited by the absorption of visible light and
relaxes by transferring electrons or electronic energy to
surrounding substrates and oxygen, exerting an unspecific toxic
effect on the cellular environment. Dodecin is a dodecameric flavin
binding protein comprising a novel ligand binding fold. It
incorporates dimers of ligands arranged in antiparallel manner
within each of the six identical binding pockets. In this thesis,
structure and function of dodecin from the archaeal organism
Halobacterium salinarum are reported. X-ray structural
investigations supplemented with functional data revealed that this
protein is an unspecific binder of flavins and binder of the
flavin-like compound lumichrome. Dissociation constants were
obtained in the nanomolar to micromolar range and found to
correlate positively with the ligand size. The preference of
dodecin for the small ligands lumichrome and lumiflavin is
described as a gated ligand binding mode, based on the low
plasticity of the dodecin binding pocket which sterically restricts
the bulkier ligands from arranging the flavin aromatic subunit in a
high affinity position. Site directed mutagenesis of the halophilic
dodecin allowed to spread the idea of dodecin as a small ligand
binding particle among homologous proteins. These mutational
studies could moreover show that the halophilic type of dodecins is
outstanding in additionally exhibiting a high affinity for
riboflavin. The stabilization of the ribityl chain by an H-bond
network to a single residue was found to suspend restrictions of
the gated ligand binding mode and to enable H. salinarum dodecin to
exhibit multiple (high) affinity. In Western-Blot and RT-PCR
analysis of the dodecin expression level, it could be demonstrated
that after a short lag period dodecin is constitutively expressed
in light and in dark. In the late stationary phase, a clear
influence of dodecin on the riboflavin cellular concentrations
could be observed. While high levels of riboflavin were found in H.
salinarum wild type cells, in cells of the dodecin deficient strain
riboflavin cellular concentrations were depressed. Lumichrome
concentrations on the other hand were unaffected from dodecin;
however; increased concentrations of lumichrome were found in
light, according to a photolytic degradation of riboflavin. In vivo
data fully agreed with the deductions from the dodecin structural
and functional investigations. Dodecin is a riboflavin binding and
carrier protein (RfBP). Its function is to store riboflavin under
non-favorable environmental conditions while preventing this flavin
from photodegradation. The lumichrome-collecting property
represents an extra-feature which allows binding of lumichrome if
degradation of riboflavin occurs in order to protect the cellular
environment from high amounts of this photo-toxic compound.