Rezümékötet 2008. - vmtdk

56
É L Õ T E R M É S Z E T T U D O M Á N Y O K
ANALYSIS OF A STRUCTURAL MOTIF IN THE
MEMBRANE-ASSOCIATED [NiFe] HYDROGENASES
Author: Emma DOROGHÁZI 3 rd year PhD student
Supervisor: Kornél L. KOVÁCS university professor;
Gábor RÁKHELY associate professor;
Gergely MARÓTI research
Institution: Department of Biotechnology, University of Szeged, Institute of Biophysics, Biological Research Center,
Hungarian Academy of Sciences, Bay-Gen Institute, Szeged
Hydrogenases, catalyzing the following reaction: 2H + + 2e ⇔ H 2, are harboured by several microorganisms. The
cells dispose of excess electrons through hydrogen production accomplished by hydrogenases, while consumption of
the molecular hydrogen mostly provides electron source for the various energy conserving processes, like respiration.
Sometimes hydrogen can be the sole energy source. Hydrogenase enzymes are distinguished by several properties. Based
on the metal content of the active centre of the hydrogenases, they are classified as [FeFe] and [NiFe] enzymes.
The core of a [NiFe] hydrogenase is composed of a large and a small subunit and they can be associated to the membrane
or localized in the cytoplasm.
Our model organism, the purple sulfur photosynthetic bacterium Thiocapsa roseopersicina contains at least four active
[NiFe] hydrogenases. The HynSL and the HupSL enzymes are attached to the cell membrane, while Hox1YH and
Hox2YH are localized in the cytoplasm.
We have noticed a highly conserved histidine-rich region with unknown function in the large subunit of [NiFe]
hydrogenases. The HxHxxHxxHxH sequence occurs in all membrane-bound hydrogenases, but only two of these
conserved histidines are present in the soluble hydrogenases.
In order to identify the function of this motif, we used site-directed mutagenesis in the His/rich region of the
Hyn hydrogenase and started to characterize the biochemical properties of the mutants. The in vivo and in vitro activity
measurements showed that the enzyme activity was affected by the replacement of these His residues but the enzyme
still remained in the membrane. Western hybridization experiment was applied to investigate the proteolytic stability
of the enzymes. It seemed that the activity differences of the mutant hydrogenases did not come from the destabilization
of the proteins.
Further investigations are in progress for getting deeper insight into the molecular mechanism of how these histidines
contribute to the biochemical properties of the hydrogenases.
Keywords: Thiocapsa roseopersicina, [NiFe] hydrogenase, histidine-rich region, site-directed mutagenesis
V I I . V A J D A S Á G I M A G Y A R T U D O M Á