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

Return to regular view	Text-only	Updated: 3/8/07