|
> Home > Faculty
& Staff > Dr. Robert Szilagyi
Structure/Function Studies of Metalloenzymes
from Thermophilic Microorganisms
The sophistication of modern theoretical levels of theory and access to computational
resources allow for using in silico approaches to investigate the
molecular details of complex biochemical processes. We are currently developing
and validating computational models and methods that expected to open up versatile
ways to probe the biochemical machinery of hyperthermophilic archaea at the
molecular level. The accuracy of these theoretical simulations is critical;
thus we utilize direct structural information from the Lawrence and Peter's
groups and spectroscopic results from the Douglas' group to fine tune the virtual
chemical models of metalloproteins and metalloenzyme. Our system of interest
is the Dps-like protein from Sulfolobus sulfataricus (SsDpsL)
as a metalloenzymatic model for oxidative stress in hyperthermophilic archaea.
We conduct a combined computational and spectroscopic investigation to determine
the molecular mechanism of peroxide decomposition that provides a molecular
model for how the chemical reactivities of acido- and thermophilic organisms
are adapted to the high oxidative stress environment. The first target area
of our research is the structural refinement of holo, apo, and partially loaded SsDpsL
metal binding sites. Contrary to other Dps proteins, the catalytically active
site is not located at the subunit-subunit interface, but buried in the core
of the four-helix bundle in a ferritin-like arrangement. This allows for construction
of small virtual chemical models without the need of addressing the undoubtedly
complex subunit-subunit interactions and periodicity of the active sites.

From a coordination chemistry perspective, the SsDpsL protein possesses
a unique active site, which can be maintained only by a highly organized network
of hydrogen bonding, dipole, and electrostatic interactions. The crystal structure
already at a modest resolution allows for virtual model building. This computational
model is being used to refine the active site structure to atomic resolution
that is currently not accessible by any means of experimental methods.
In the second target area, we are probing two prototypical reaction mechanisms
that have been characterized so far for ferritin-like protein cages:
Perodixase:
[MIIMIIHis2(OOCR)4]
+ H2O2 + 2 H+ → [MIII-OH2-MIIIHis2(OOCR)4] + H2O
[MIII-OH2-MIIIHis2(OOCR)4]
+ 2 e- → [MIIMIIHis2(OOCR)4] + H2O
Catalase:
[MIIMIIHis2(OOCR)4]
+ H2O2 + 2 H+ →[MIII-O-MIIIHis2(OOCR)4] + H2O
[MIII-O-MIIIHis2(OOCR)4]
+ H2O2 →[MIIMIIHis2(OOCR)4] + O2 + H2O
Using the quantum chemically refined active site and magnetic coupling constant
measurements from the Douglas' lab and their collaborators, we are in the position
to construct a close to 150-atom functional model of the manganese bound SsDpsL
active site. This model is being validated by metal binding, peroxide uptake
and dioxygen evolution assays that are being carried out parallel to the computational
studies.
Current Laboratory Personnel:
David Mulder, Ph.D. Student
Tyler Arbour, Undergraduate Student
Jessica Wittmann, Undergraduate Student

Jessica Whitman and Robert Szilagyi in the Szilagyi lab
|